A newly discovered human bocavirus, HBoV - a member of the parvovirus family - appears to be a significant respiratory pathogen prevalent in children. This application proposes a collaborative effort to further characterize this virus by a molecular virology group with extensive parvovirus research expertise and a pediatric infectious disease group with a proven track record of discovery in pediatric virology and epidemiology. We have identified several pediatric samples that contain the virus, as detected by diagnostic PCR, and have cloned the gene for the predicted major viral capsid protein from these samples, into a baculovirus vector. Infection of insect cells with this baculovirus results in expression of the bocaviral polypeptide and its efficient assembly into virus-like particles (VLPs). VLPs will be produced in the quantities necessary to produce HboV capsid-specific polyclonal and monoclonal antibodies, and to develop an ELISA for the detection of anti-capsid IgG and IgM antibodies. A combination of PCR and ELISA will be used, in both prospective and retrospective studies, to explore the distribution and disease associations of HBoV in the human population, particularly within our large pediatric study group. We will attempt to grow the virus and study its biology in cell culture. For this we will produce antibodies able to detect putative viral non-structural gene products expressed in HBoV infected cell, and establish an infected-cell culture system for the closely- related bovine parvovirus (BPV-1), in order to validate these reagents. We will inoculate several potentially appropriate human cell lines, such as embryonic lung fibroblasts, non-small cell lung cancer, SV40- transformed lung epithelial cells and stepwise immortalized and transformed airway epithelial cells. These will be examined for their ability to support single- and multiple-round infections with HoBV, using the serological reagents generated as above. Once we have identified a cell line capable of expanding HBoV in culture, we will purify viral genomes from virions grown in such cells, construct a full-length clone of the virus by in vitro DNA replication, and test its infectivity following transfection back into competent host cells in vitro. These studies are designed to develop new tools for the detection and isolation of the newly-discovered human bocavirus (HboV), and to identify antibodies to this virus in human serum. These tools will be used to establish the basic epidemiology of HboV and to explore its role in respiratory illness among infants. [unreadable] [unreadable] [unreadable]